The invention relates to a method for confocal 4-pi microscopy.
The invention furthermore relates to a confocal 4-pi microscope with a light source that produces an illumination light that exhibits at least one illumination wavelength, and that is directable from two sides by one objective each onto a sample, whereby a stationary illumination wave having a main illumination maximum and secondary illumination maxima is produced by interference of the illumination light in the sample, and having a detector to detect detection light emitted by the sample that passes through both objectives and that exhibits at least one detection wavelength.
A sample is illuminated with a light beam in scanning microscopy in order to observe the reflected or fluorescent light emitted by the sample. The focus of an illumination light beam is moved in an object plane with the help of a controllable beam deflector, generally by tilting two mirrors, in which case the deflection axes are most often perpendicular to each other so that one mirror deflects in x-direction and the other in y-direction. The mirrors may, for example, be tilted with the help of galvanometric positioners. The power of the light coming from the object is measured, depending on the position of the scanning beam. Generally, the positioners are equipped with sensors to determine the current position of the mirror.
In confocal scanning microscopy specifically, an object is scanned in three dimensions with the focus of a light beam. In general, a confocal scanning microscope comprises a light source, a focusing optic with which the light from the light source is focused on a pinhole aperture—the so-called excitation aperture—, a beam splitter, a beam deflector to control the beam, a microscope optic, a detection aperture, and detectors to detect the detection and/or fluorescent light. The illumination light is coupled by a beam splitter. The detection light emitted by the object, such as fluorescent or reflected light or even CARS light, returns to the beam splitter via the beam deflector, passes through it, and is finally focused on the detection aperture, behind which are located the detectors. Detection light that does not originate directly from the focus region takes another light path and does not pass through the detection aperture, so that one obtains point information that results in a three-dimensional image when the object is scanned sequentially. Most often, a three-dimensional image is achieved by taking layers of image data, in which case the path of the scanning beam ideally describes a meandering pattern on or in the object. (Scanning a line in x-direction at a constant y-position, then continuing x-scanning and by y-shifting to the next line to be scanned, scanning this line in a negative x-direction at a constant y-position, etc.). To enable layered data imaging, the sample table or the objective is shifted after scanning one layer so that the next layer to be scanned is brought into the focal plane of the objective.
Increasing resolution in the direction of the optical axis may be achieved by a double-objective arrangement as described in European patent EP 0 491 289 with the title “Double-confocal scanning microscope.” The light coming from the illumination system is split into two partial beams that illuminate the sample simultaneously in that they run in opposite directions to each other through two objectives that are arranged in mirror symmetry. The two objectives are arranged on different sides of their common object plane. An interference pattern, which exhibits one main maximum and several secondary maxima during constructive interference, is formed in the object point as a result of this interferometric illumination. Here, the secondary maxima are generally arranged along the optical axis. Increased axial resolution can be achieved with a double-confocal scanning microscope in comparison to a conventional scanning microscope by interferometric illumination.
The secondary maxima produce disturbing double images during imaging, which must be removed using mathematical image reconstruction methods. The lower the secondary maxima, the better these methods work. It has been shown in practice that artifact-free reconstruction can be achieved only when the amplitudes of the secondary maxima are at most 50% of the amplitude of the main maximum. However, the single-photon excitation preferred in confocal microscopy generates secondary maxima of approximately 65% level in a standard 4-pi microscope and therefore cannot be used for imaging requiring structural resolution.
A method and a device to illuminate a transparent object, especially for use in the double-confocal scanning microscopy is known from published German patent application DE 100 10 154 A1, in which to illuminate a point of the object two light waves running in opposite directions from a coherent light source (focused on the point) interfere creating an illumination pattern, and in which avoidance of the problem of reconstruction is characterized in that at least two additional converging coherent light waves overlap in order to minimize the secondary maxima of the illumination pattern.
A double confocal scanning microscope with an illumination beam path, a light source, and a detection beam path of a detector is known from published German patent application DE 101 07 095 A1. The double confocal scanning microscope is characterized in that to avoid the cause of the problem of the reconstruction methods at least one optical component is provided that acts on the illumination and/or detection beam path and is configured such that it affects the amplitude and/or the phase and/or the polarization of the light, thereby making changeable the characteristics of the double confocal illumination and/or detection.